The following discussion of the background and of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
The present invention relates to gene delivery and gene therapy, and provides novel nucleic acid constructs for expression of human IL-2 in a mammal, formulations for delivery that incorporate a nucleic acid construct for expression, and methods for preparing and using such constructs and formulations. In particular, this invention relates to plasmid constructs for delivery of therapeutic IL-2 encoding nucleic acids to cells and to modulation of cytokine activity. In addition, this invention relates to methods of using those constructs as well as methods for preparing such constructs.
Plasmids are an essential element in genetic engineering and gene therapy. Plasmids are circular DNA molecules that can be introduced into bacterial cells by transformation which replicate autonomously in the cell. Plasmids allow for the amplification of cloned DNA. Some plasmids are present in 20 to 50 copies during cell growth, and after the arrest of protein synthesis, as many as 1000 copies per cell of a plasmid can be generated. (Suzuki et al., Genetic Analysis, p. 404, (1989).)
Current non-viral approaches to human gene therapy require that a potential therapeutic gene be cloned into plasmids. Large quantities of a bacterial host harboring the plasmid may be fermented and the plasmid DNA may be purified for subsequent use. Current human clinical trials using plasmids utilize this approach. (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6: 535-548). Studies normally focus on the therapeutic gene and the elements that control its expression in the patient when designing and constructing gene therapy plasmids. Generally, therapeutic genes and regulatory elements are simply inserted into existing cloning vectors that are convenient and readily available.
Plasmid design and construction utilizes several key factors. First, plasmid replication origins determine plasmid copy number, which affects production yields. Plasmids that replicate to higher copy number can increase plasmid yield from a given volume of culture, but excessive copy number can be deleterious to the bacteria and lead to undesirable effects (Fitzwater, et al., Embo J. 7: 3289-3297 (1988); Uhlin, et al., Mol. Gen. Genet. 165: 167-179 (1979)). Artificially constructed plasmids are sometimes unstably maintained, leading to accumulation of plasmid-free cells and reduced production yields.
To overcome this problem of plasmid-free cells, genes that code for antibiotic resistance phenotype are included on the plasmid and antibiotics are added to kill or inhibit plasmid-free cells. Most general purpose cloning vectors contain ampicillin resistance (.beta.-lactamase, or bla) genes. Use of ampicillin can be problematic because of the potential for residual antibiotic in the purified DNA, which can cause an allergic reaction in a treated patient. In addition, .beta.-lactam antibiotics are clinically important for disease treatment. When plasmids containing antibiotic resistance genes are used, the potential exists for the transfer of antibiotic resistance genes to a potential pathogen.
Other studies have used the neo gene which is derived from the bacterial transposon Tn5. The neo gene encodes resistance to kanamycin and neomycin (Smith, Vaccine 12: 1515-1519 (1994)). This gene has been used in a number of gene therapy studies, including several human clinical trials (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6: 535-548). Due to the mechanism by which resistance is imparted, residual antibiotic and transmission of the gene to potential pathogens may be less of a problem than for .beta.-lactams.
In addition to elements that affect the behavior of the plasmid within the host bacteria, such as E. coli, plasmid vectors have also been shown to affect transfection and expression in eukaryotic cells. Certain plasmid sequences have been shown to reduce expression of eukaryotic genes in eukaryotic cells when carried in cis (Peterson, et al., Mol. Cell. Biol. 7: 1563-1567 (1987); Yoder and Ganesan, Mol. Cell. Biol. 3: 956-959 (1983); Lusky and Botchan, Nature 293: 79-81 (1981); and Leite, et al., Gene 82: 351-356 (1989)). Plasmid sequences also have been shown to fortuitously contain binding sites for transcriptional control proteins (Ghersa, et al., Gene 151: 331-332 (1994); Tully and Cidlowski, Biochem. Biophys. Res. Comm. 144: 1-10 (1987); and Kushner, et al., Mol. Endocrinol. 8: 405-407 (1994)). This can cause inappropriate levels of gene expression in treated patients.
Interleukin-2 (IL-2) is, among other functions, involved in stimulating the proliferation of helper T cells, in particular T.sub.H 1 cells by an autocrine mechanism. Secretion of IL-2 will also stimulate the proliferation of other activated helper T cells and cytotoxic T cells.
Based on these and other responses to IL-2, attempts have been made to use IL-2 in anti-tumor therapy. IL-2 polypeptide can be isolated from stimulated T.sub.H 1 cells or produced from a recombinant IL-2 gene. Such a recombinant gene is described, for example, in Taniguchi et al., U.S. Pat. No. 4,738,927, along with cells containing the recombinant gene. However, administration of high doses of IL-2 polypeptide results in significant toxicity-related side effects, such as fever, fluid retention, and vascular leak syndrome. In addition, administration of polypeptide must be repeated at short intervals due to a short half-life for the injected polypeptide.
As an example of an alternative, Hobart et al., PCT application PCT/US95/15020, International publication WO 96/17063, describes a plasmid encoding human IL-2, in which a natural human IL-2 coding sequence is part of an expression facilitating sequence which also contains a cytomegalovirus (CMV) immediate-early promoter region and a transcription termination/polyadenylation signal sequence derived from bovine growth hormone. Formulations for delivery of human IL-2 coding sequence are also described which included the IL-2 encoding plasmid with DMRIE/DOPE or .beta.AE/DOPE at a DNA:lipid mass ratio of 5:1.